Rates of thyroid cancer have tripled in the last few decades, making it the fastest growing form of cancer. 80% of thyroid cancer cases are Papillary Thyroid Carcinoma (PTC), which arises from transformation of follicular cells in the thyroid. Metastatic PTC has no effective treatment, a five-year survival rate of 50%, and results in the death of approximately 1500 patients in the US each year. 5-30% of PTC cases involve fusion proteins in which the Ret receptor tyrosine kinase is fused to the coiled-coiled domain of various cytoplasmic proteins. Recently, Ret fusions have also been identified in a subset of lung adenocarcinomas. Thus, therapeutics for Ret fusion-driven tumors remains an unmet need. Two Ret fusions, CCDC6-RET (PTC1) and NcoA4-RET (PTC3) account for greater than 90% of fusions found in PTC. Although both fusion proteins lead to activation of Ret, the two fusions are associated with different PTC subtypes. CCDC6-RET is closely associated with the classic variant, a more benign subtype, and NcoA4-RET is closely associated with the solid subtype, which is more aggressive and malignant. Work by the Cagan lab has validated Drosophila models of oncogenic Ret isoforms that are associated with Medullary Thyroid Carcinoma (MTC). These models were used to explore function as well as identify lead therapeutic hits. The aim of this proposal is to characterize the signaling pathways activated by Ret fusions CCDC6-RET and NcoA4-RET, identify differences that are clinically significant, and develop an optimized therapy. First, signaling changes caused by expression of CCDC6-RET and NcoA4-RET in Drosophila will be characterized and compared by Western blot analysis and immunoflourescence. Secondly, clinically relevant kinase inhibitors will be fed to flies expressing either Ret fusion to identify potential therapies based on rescue of transgene-induced phenotypes. Lastly, the relevance of genetic complexity of PTC tumors on treatment will be evaluated using a functional driver vs. passenger screen using a pre-existing Drosophila kinome RNAi library. All identified therapies will be evaluated in mammalian models, such as cancer cell lines, to ensure the findings are relevant to patients. The innovative studies proposed here will provide critical insight into how Ret fusions drive PTC, how different Ret fusions may differ in their effects, and how Ret fusion driven tumors may respond to targeted therapies.